Soybean Transgenic Event MON87705 and Methods for Detection Thereof

ABSTRACT

The present invention provides a transgenic soybean event MON87705, and cells, seeds, and plants comprising DNA diagnostic for the soybean event. The invention also provides compositions comprising nucleotide sequences that are diagnostic for said soybean event in a sample, methods for detecting the presence of said soybean event nucleotide sequences in a sample, probes and primers for use in detecting nucleotide sequences that are diagnostic for the presence of said soybean event in a sample, growing the seeds of such soybean event into soybean plants, and breeding to produce soybean plants comprising DNA diagnostic for the soybean event.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/100,859, filed on Sep. 29, 2008.

INCORPORATION OF SEQUENCE LISTING

This application contains an electronic equivalent paper copy of thesequence listing submitted herewith electronically via EFS web and acomputer-readable form of the sequence listing submitted herewithelectronically via EFS web which contains a file named“56047-0001US_seqlist.txt”, which is 50,960 bytes in size (measured inMS-DOS) and which was created on Sep. 8, 2009. The sequence listingssubmitted herewith as electronic equivalents of a paper copy andcomputer-readable form are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to transgenic soybean event MON87705 andplant parts and seed thereof. The event exhibits an oil compositioncomprising altered fatty acid levels. The invention also relates tomethods for detecting the presence of soybean event in a biologicalsample, and provides nucleotide sequences that are capable of doing so.

BACKGROUND OF THE INVENTION

Soybean is an important crop and is a primary food source in many areasof the world. The methods of biotechnology have been applied to soybeanfor improvement of agronomic traits and the quality of the product. Onesuch quality trait is a soybean oil comprising altered fatty acidlevels.

It would be advantageous to be able to detect the presence oftransgene/genomic DNA of a particular plant in order to determinewhether progeny of a sexual cross contain the transgene/genomic DNA ofinterest. In addition, a method for detecting a particular plant wouldbe helpful when complying with regulations requiring the pre-marketapproval and labeling of foods derived from the recombinant crop plants.

Soybean oils have been modified by various breeding methods to createbenefits for specific markets. However, a soybean oil that is broadlybeneficial to major soybean oil users such as consumers of salad oil,cooking oil and frying oil, and industrial markets such as biodiesel andbiolube markets, is not available. Prior soybean oils were either tooexpensive or lacked an important food quality property such as oxidativestability, good fried food flavor or saturated fat content or animportant biodiesel property such as appropriate nitric oxide emissionsor cold tolerance or cold flow.

Soybean oil typically contains approximately 20% oleic acid. Oleic acidhas one double bond, but is still relatively stable at hightemperatures, and oils with high levels of oleic acid are suitable forcooking and other processes where heating is required. Recently,increased consumption of high oleic oils has been recommended, becauseoleic acid appears to lower blood levels of low density lipoproteins(“LDLs”) without affecting levels of high density lipoproteins (“HDLs”).However, some limitation of oleic acid levels is desirable, because whenoleic acid is degraded at high temperatures, it creates negative flavorcompounds and diminishes the positive flavors created by the oxidationof linoleic acid. Neff et al., JAOCS, 77:1303-1313 (2000); Warner etal., J. Agric. Food Chem. 49:899-905 (2001). It is thus preferable touse oils with oleic acid levels that are 65-85% or less by weight, inorder to limit off-flavors in food applications such as frying oil andfried food. Other preferred oils have oleic acid levels that are greaterthan 55% by weight in order to improve oxidative stability.

For many oil applications, saturated fatty acid levels of less than 8%by weight or even less than about 2-3% by weight are desirable. Soybeanoil typically contains about 16-20% saturated fatty acids: 13-16%palmitate and 3-4% stearate (see generally Gunstone et al., The LipidHandbook, Chapman & Hall, London (1994)). Saturated fatty acids havehigh melting points which are undesirable in many applications. Whenused as a feedstock or fuel, saturated fatty acids cause clouding at lowtemperatures and confer poor cold flow properties such as pour pointsand cold filter plugging points to the fuel. Oil products containing lowsaturated fatty acid levels may be preferred by consumers and the foodindustry because they are perceived as healthier and/or may be labeledas “saturated fat free” in accordance with FDA guidelines. In addition,low saturate oils reduce or eliminate the need to winterize the oil forfood applications such as salad oils. In biodiesel and lubricantapplications oils with low saturated fatty acid levels confer improvedcold flow properties and do not cloud at low temperatures.

Soybean lines that produce seed with mid-oleic and low saturate contentwould be desirable. Methods disclosed here enable production of soybeanseeds that have oleic acid levels in the mid oleic range of 55-80% andsaturated fatty acid levels of less than 8%.

The expression of foreign genes in plants is known to be influenced bytheir chromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., 1988Ann. Rev. Genet 22:421-477). For this reason, it is often necessary toscreen a large number of events in order to identify an eventcharacterized by optimal expression of an introduced gene of interest.For example, it has been observed in plants and in other organisms thatthere may be wide variation in the levels of expression of an introducedgene among events. There may also be differences in spatial or temporalpatterns of expression, for example, differences in the relativeexpression of a transgene in various plant tissues, that may notcorrespond to the patterns expected from transcriptional regulatoryelements present in the introduced gene construct. For this reason, itis common to produce several hundred to several thousand differentevents and screen the events for a single event that has the desiredtransgene expression levels and patterns for commercial purposes. Anevent that has the desired levels or patterns of transgene expression isuseful for introgressing the transgene into other genetic backgrounds bysexual outcrossing using conventional breeding methods. Progeny of suchcrosses maintain the transgene expression characteristics of theoriginal transformant. This strategy is used to ensure reliable geneexpression in a number of varieties that are suitably adapted tospecific local growing conditions.

It is possible to detect the presence of a transgene by any well knownnucleic acid detection method such as the polymerase chain reaction(PCR) or DNA hybridization using nucleic acid probes. These detectionmethods generally focus on frequently used genetic elements, such aspromoters, terminators, marker genes, etc. As a result, such methods maynot be useful for discriminating between different events, particularlythose produced using the same DNA construct unless the sequence ofchromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known.An event-specific PCR assay is discussed, for example, by Taverniers etal. (J. Agric. Food Chem., 53: 3041-3052, 2005) in which anevent-specific tracing system for transgenic maize lines Bt11, Bt176,and GA21 and for canola event GT73 is demonstrated. In this study,event-specific primers and probes were designed based upon the sequencesof the genome/transgene junctions for each event. Transgenic plantevent-specific DNA detection methods have also been described in U.S.Pat. Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880;6,900,014 and 6,818,807.

This invention relates to the transgenic soybean (Glycine max) plantMON87705 with an oil composition comprising altered fatty acid levelsand to the DNA construct of soybean plant MON87705 and the detection ofthe transgene/genomic insertion region in soybean MON87705 and progenythereof.

SUMMARY OF THE INVENTION

The present invention includes a transgenic soybean plant designatedMON87705 and progeny that are indistinguishable from soybean eventMON87705 (to the extent that such progeny also contain at least oneallele that corresponds to the inserted transgenic DNA) thereof havingseed deposited with American Type Culture Collection (ATCC) withAccession No. PTA-9241. Another aspect of the invention is the progenyplants, or seeds, or regenerable parts of the plants and seeds of thesoybean event MON87705. The invention also includes plant parts of thesoybean event MON87705 that include, but are not limited to pollen,ovule, flowers, shoots, roots, stems, leaves, pods, seeds andmeristematic tissues. Novel genetic compositions contained in the genomeof MON87705 and products from MON87705 such as oil, meal, flour, foodproducts, protein supplements and biomasses remaining in a field fromwhich soybean plants corresponding to MON87705 have been harvested areaspects of this invention.

The invention provides a soybean plant capable of producing a seedhaving an oil composition comprising approximately 55-80% oleic acid byweight and less than 8% saturated fatty acid by weight, wherein geneticdeterminants for said oil composition is obtainable from soybean havingATCC Accession No. PTA-9241.

According to one aspect of the invention, compositions and methods areprovided for detecting the presence of the transgene/genomic insertionregion from a novel soybean plant designated MON87705. DNA sequences areprovided that comprise at least one junction sequence of MON87705selected from the group consisting of SEQ ID NO: 1 ([A] corresponding topositions 3449 through 3468 of SEQ ID NO: 6 [F], FIG. 2), SEQ ID NO: 2([B] corresponding to positions 10700 through 10719 of SEQ ID NO: 6 [F],FIG. 2) and SEQ ID NO: 18 (corresponding to positions 9266 through 9371of SEQ ID NO: 6 [F], FIG. 2) and complements thereof wherein a junctionsequence is a nucleotide sequence that spans the point at whichheterologous DNA inserted into the genome is linked to the soybean cellgenomic DNA and detection of this sequence in a biological samplecontaining soybean DNA is diagnostic for the presence of the soy eventMON87705 DNA in said sample (FIG. 2). Such junction sequences cancontain at least one of the sequences listed under SEQ ID NO: 1, 2, 18and the complements thereof. A soybean plant, soybean seed from theplant and progeny of the plant comprising these DNA molecules is anaspect of this invention.

DNA sequences that comprise novel transgene/genomic insertion region,SEQ ID NO: 3 [C], SEQ ID NO: 4 [D] and SEQ ID NO: 5 [E] or SEQ ID NO: 1[A] and SEQ ID NO: 2 [B] (see FIG. 2) from soybean event MON87705 areaspects of this invention. The soybean plant, seed and progenycomprising these molecules are also aspects of this invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, where the first DNA moleculecomprises at least 11 or more, at least 12 or more, or at least 13 ormore contiguous polynucleotides of any portion of the transgene regionof the DNA molecule of SEQ ID NO: 5 and a DNA molecule of similar lengthof any portion of a 5′ flanking soybean genomic DNA region of SEQ ID NO:3, where these DNA molecules when used together are useful as DNAprimers in a DNA amplification method that produces an amplicon. Theamplicon produced using these DNA primers in the DNA amplificationmethod is diagnostic for soybean event MON87705 when the ampliconcontains SEQ ID NO: 1. Any amplicon produced by DNA primers homologousor complementary to any portion of SEQ ID NO: 3 and SEQ ID NO: 5, andany amplicon that comprises SEQ ID NO: 1 is an aspect of the invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO: 5 anda DNA molecule of similar length of any portion of a 3′ flanking soybeangenomic DNA of SEQ ID NO: 4, where these DNA molecules are useful as DNAprimers in a DNA amplification method. The amplicon produced using theseDNA primers in the DNA amplification method is diagnostic for soybeanevent MON87705 when the amplicon contains SEQ ID NO: 2. Any ampliconsproduced by DNA primers homologous or complementary to any portion ofSEQ ID NO: 4 and SEQ ID NO: 5, and any amplicon that comprises SEQ IDNO: 2 is an aspect of the invention.

According to another aspect of the invention, two DNA molecules areprovided for use in a DNA detection method, wherein the first DNAmolecule comprises at least 11 or more contiguous polynucleotides of anyportion of the transgene region of the DNA molecule of SEQ ID NO: 5 ortheir complements wherein one primer is derived from sequence 5′ to SEQID NO: 18 and the other primer is derived from sequence 3′ to SEQ ID NO:18 where these DNA molecules are useful as DNA primers in a DNAamplification method. The amplicon produced using these DNA primers inthe DNA amplification method is diagnostic for soybean event MON87705when the amplicon contains SEQ ID NO: 18. Any amplicons produced by DNAprimers homologous or complementary to any portion of SEQ ID NO: 5 andany amplicon that comprises SEQ ID NO: 18 is an aspect of the invention.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to the soybean event MON87705 in a sampleare provided. Such methods comprise: (a) contacting the samplecomprising DNA with a primer set that, when used in a nucleic acidamplification reaction with genomic DNA from soybean event MON87705,produces an amplicon that is diagnostic for soybean event MON87705; (b)performing a nucleic acid amplification reaction, thereby producing theamplicon; and (c) detecting the amplicon wherein said amplicon comprisesone or more or the sequences listed under SEQ ID NOs: 1, 2 and 18.

Another aspect of the invention is a soybean plant, or seed, or productcontaining the event of the plant or seed of MON87705 wherein thegenomic DNA comprises a DNA molecule consisting essentially of thenucleotide sequence of SEQ ID NO: 3 from about positions 1 to 3458, thenucleotide sequence of SEQ ID NO: 5 from about positions 1 to 7251 andthe nucleotide sequence of SEQ ID NO: 4 from about positions 1 to 2515(the contig of which is presented as SEQ ID NO: 6), and complementsthereof. A further aspect of the invention is a soybean plant, or seed,or product derived from the plant or seed of MON87705 wherein thegenomic DNA comprises a DNA molecule consisting essentially of thenucleotide sequence of SEQ ID NO: 6 from about positions 1 to 13224 andcomplements thereof. A soybean plant, or seed, or product derived fromthe plant or seed MON87705, in which the genomic DNA when isolated fromthe soybean plant, or seed, or product comprises a DNA moleculeincorporating SEQ ID NOs: 1, 2 or 18, and complements thereof. In oneaspect of the invention, the DNA molecule contains the soybean eventMON87705. In another aspect, two copies of the DNA molecule containingthe soybean event MON87705 are present in the soybean plant. In anotheraspect, one copy of the DNA molecule containing the soybean eventMON87705 is present in the soybean plant.

Another aspect of the invention is a soybean plant, or seed, or productcontaining the event of the plant or seed of MON87705, in which thegenomic DNA when isolated from the soybean plant, or seed, or productproduces an amplicon in a DNA amplification method, where said ampliconcomprises at least one sequence from the group consisting of SEQ ID NOs:1, 2 and 18.

In another aspect, seeds of the soybean plants may be placed in acontainer. As used herein, a container is any object capable of holdingsuch seeds. A container preferably contains greater than about 500,1,000, 5,000, or 25,000 seeds where at least about 10%, 25%, 50%, 75% or100% of the seeds are derived from a plant of the present invention. Thepresent invention also provides a container of over about 10,000, morepreferably about 20,000, and even more preferably about 40,000 seedswhere over about 10%, more preferably about 25%, more preferably 50% andeven more preferably about 75% or 90% of the seeds are seeds derivedfrom a plant of the present invention. The present invention alsoprovides a container of over about 10 kg, more preferably about 25 kg,and even more preferably about 50 kg seeds where over about 10%, morepreferably about 25%, more preferably about 50% and even more preferablyabout 75% or 90% of the seeds are seeds derived from a plant of thepresent invention.

Another aspect of the invention provides a method for detecting thepresence or absence of soybean transgenic event MON87705 in a biologicalsample comprising a) extracting DNA from said sample; and b) assayingthe presence or absence of a polynucleotide having a sequencecorresponding to that shown under SEQ ID NOs: 1, 2 or 18, whereby thepresence or absence of soybean event MON87705 in said sample can beascertained.

According to another aspect of the invention, methods of detecting thepresence of DNA corresponding to the MON87705 event in a sample,comprising: (a) contacting the sample with a probe that hybridizes understringent hybridization conditions with a sequence selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and theircomplements, and does not hybridize under the stringent hybridizationconditions with soybean plant DNA that does not comprise SEQ ID NO: 1and SEQ ID NO: 2; (b) subjecting the sample and probe to stringenthybridization conditions; and (c) detecting binding of the probe to saidsample; wherein binding is diagnostic for the presence of said DNA insaid sample. Another aspect of the invention is a probe comprising fromabout 11- to about 20 consecutive nucleotides in length for use indetecting the presence of soybean event MON87705 in a biological sample,wherein said consecutive nucleotides are selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2, and their complements. Theprobe may be a deoxyribonucleic acid, a ribonucleic acid or a nucleotideanalogue. The probe may be labeled with at least one fluorophore.

Another aspect of the invention is a method of determining zygosity ofthe progeny of soybean event MON87705, the method comprising (a)contacting the sample comprising soybean DNA with the primer set SQ21928(SEQ ID NO: 11), SQ20901 (SEQ ID NO: 12), and the probe 6FAM™-labeledPB10164 (SEQ ID NO: 13) and that when used in a nucleic-acidamplification reaction with genomic DNA from soybean event MON87705,produces a first amplicon, releasing a fluorescent signal from thecombination of primers SQ21928 and SQ20901 and a 6FAM™-labeledprimer/probe, PB10164 that is diagnostic for soybean event MON87705 (b)performing a nucleic acid amplification reaction, thereby producing thefirst amplicon; and (c) detecting said first amplicon; and (d)contacting the sample comprising soybean DNA with the primer set,SQ21928 (SEQ ID NO: 11) and SQ21905 (SEQ ID NO: 15), and a VIC™-labeledPB10335 (SEQ ID NO: 14) that when used in a nucleic-acid amplificationreaction with genomic DNA from soybean plants produces a secondamplicon, releasing a fluorescent signal that is diagnostic of thewild-type soybean genomic DNA homologous to the soybean genomic regionof a transgene insertion identified as soybean event MON87705; (e)performing a nucleic acid amplification reaction, thereby producing thesecond amplicon and (f) detecting said second amplicon; and (g)comparing the first and second amplicons in a sample, wherein thepresence of both amplicons indicates the sample is heterozygous for thetransgene insertion.

Another aspect of the invention is a method of determining zygosity ofthe progeny of soybean event MON87705, the method comprising (a)contacting the sample comprising soybean DNA with the primer set SQ21928(SEQ ID NO: 11), SQ20901 (SEQ ID NO: 12), and SQ21905 (SEQ ID NO: 15),that when used in a nucleic-acid amplification reaction with genomic DNAfrom soybean event MON87705, produces a first amplicon from thecombination of primers SQ21928 and SQ20901 that is diagnostic forsoybean event MON87705 (b) performing a nucleic acid amplificationreaction, thereby producing the first amplicon; and (c) detecting saidfirst amplicon; and (d) contacting the sample comprising soybean DNAwith the primer set, SQ21928 and SQ21905, that when used in anucleic-acid amplification reaction with genomic DNA from soybean plantsproduces a second amplicon from the combination of primers SQ21928 andSQ21905 that is diagnostic of the wild-type soybean genomic DNAhomologous to the soybean genomic region of a transgene insertionidentified as soybean event MON87705; (e) performing a nucleic acidamplification reaction, thereby producing the second amplicon and (f)detecting said second amplicon; and (g) comparing the first and secondamplicons in a sample, wherein the presence of both amplicons indicatesthe sample is heterozygous for the transgene insertion.

The invention also provides a composition having a DNA molecule selectedfrom the group consisting of SEQ ID NOs: 1, 2 and 18, wherein saidcomposition is a commodity product selected from the group consisting ofsoybean meal, soy flour, soy protein concentrate, soy protein isolates,texturized soy protein concentrate, hydrolyzed soy protein and whippedtopping.

Another aspect of the invention is a method for detecting the presenceof a nucleotide sequence diagnostic for the presence of soybean eventMON87705 in a biological sample, comprising detecting the presence of anucleotide sequence wherein said sequence is selected from the groupconsisting of SEQ ID NOs: 1, 2 and 18, wherein said biological sample isselected from the group consisting of soybean meal, soy flour, soyprotein concentrate, soy protein isolates, texturized soy proteinconcentrate, hydrolyzed soy protein and whipped topping.

Kits for the detection of soybean event MON87705 in a soybean sample areprovided that comprise nucleotide components designed based on detectingone or more sequences shown under SEQ ID NOs: 1, 2 and 18. Kits for thedetection of soybean event MON87705 are provided which use primersdesigned from SEQ ID NO: 3, 4 or 5. An amplicon produced using said kitis diagnostic for MON87705 when the amplicon contains one or morenucleotide sequences listed under SEQ ID NOs: 1, 2 and 18.

Another aspect of the invention is a method for producing a soybeanplant comprising altered fatty acid levels, comprising crossing a plantcomprising soybean event MON87705 with a soybean plant lacking soybeanevent MON87705 to obtain a plant comprising said soybean event MON87705and altered fatty acid levels, wherein a representative sample of seedcomprising said event was deposited under ATCC Accession No. PTA-9241.

Also provided is a method of producing a soybean variety comprisingsoybean event MON87705, comprising backcrossing soybean event MON87769into said variety, wherein a representative sample of seed comprisingsaid event was deposited under ATCC Accession No. PTA-9241.

Another aspect of the invention is a soybean plant capable of producingseeds having an oil composition comprising approx. 55-80% oleic acid andless than 8% saturated fatty acid, wherein genetic determinants for saidoil composition is obtainable from soybean having ATCC Accession No.PTA-9241. Also provided is an oil composition obtained from seeds of thesoybean plant and a commodity product derived from the oil selected fromthe group consisting of cooking oil, salad oil, shortening, lecithin,nontoxic plastics, printing inks, lubricants, waxes, hydraulic fluids,electric transformer fluids, solvents, cosmetics, hair care products andbiodiesel.

An oil of the present invention may be blended with other oils. In oneaspect, the oil produced from plants of the present invention orgenerated by a method of the present invention constitutes greater than0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weight of the oilcomponent of any product. In another aspect, the oil preparation may beblended and can constitute greater than 10%, 25%, 35%, 50% or 75% of theblend by volume. Oil produced from a plant of the present invention canbe admixed with one or more organic solvents or petroleum distillates.

A further aspect of the invention is a genome of a soybean cellcomprising a polynucleotide having a sequence selected from the groupconsisting of sequences listed under SEQ ID NO: 1, 2 and 18.

Another aspect of the invention is a soybean plant, or seed, or seedprogeny, or product derived from the plant or seed of MON87705. Seed forsale for planting or for making commodity products is an aspect of theinvention. Such commodity products include, but are not limited to,whole or processed soy seeds, animal feed, vegetable oil, meal, flour,nontoxic plastics, printing inks, lubricants, waxes, hydraulic fluids,electric transformer fluids, solvents, cosmetics, hair care products,soymilk, soy nut butter, natto, tempeh, soy protein concentrate, soyprotein isolates, texturized soy protein concentrate, hydrolyzed soyprotein, whipped topping, cooking oil, salad oil, shortening, lecithin,edible whole soybeans (raw, roasted, or as edamamé), soymilk, soyyogurt, soy cheese, tofu, yuba and biodiesel.

The foregoing and other aspects of the invention will become moreapparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Map of binary transformation vector, pMON95829 that was used togenerate soybean plant MON87705.

FIG. 2. Organization of the transgenic insert in the genome of soybeanevent MON87705; [A] corresponds to the relative position of SEQ ID NO: 1which forms the junction between SEQ ID NO: 3 and SEQ ID NO: 5; [B]corresponds to the relative position of SEQ ID NO: 2 which forms thejunction between SEQ ID NO: 4 and SEQ ID NO: 5; [C] corresponds to therelative position of SEQ ID NO: 3, the soybean genome sequence flankingthe arbitrarily assigned/designated 5′ end of the expression cassetteintegrated into the genome in event MON87705; [D] corresponds to therelative position of SEQ ID NO: 4, the soybean genome sequence flankingthe arbitrarily assigned/designated 3′ end of the expression cassetteintegrated into the genome in event MON87705; [E] represents the variouselements comprising SEQ ID NO: 5 and is the sequence of the expressioncassette inserted into the genome of the event MON87705; and [F]represents the contiguous sequence comprising, as represented in thefigure from left to right, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:4, inwhich SEQ ID NO:1 and SEQ ID NO:2 are incorporated as set forth above,as these sequences are present in the genome in event MON87705.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—A 20 nucleotide sequence representing the 5′ left borderjunction between the soybean genomic DNA and the integrated T-DNA. Thissequence corresponds to positions 3413 to 3432 of SEQ ID NO: 6. Inaddition, SEQ ID NO: 1 ([A] of FIG. 2) is a nucleotide sequencecorresponding to positions 3432 through 3422 of SEQ ID NO: 3 ([C], seeFIG. 2) and the integrated 5′ left border of the integrated T-DNAcorresponding to positions 1 through 10 of SEQ ID NO: 5 ([E], see FIG.2).SEQ ID NO: 2—A 20 nucleotide sequence representing the 3′ left borderjunction between the integrated T-DNA and the soybean genomic DNA. Thissequence corresponds to positions 10664 to 10683 of SEQ ID NO: 6. Inaddition, SEQ ID NO: 2 ([B], see FIG. 2) is a nucleotide sequencecorresponding positions 7242 through 7251 of SEQ ID NO: 5 ([E], see FIG.2) and the 3′ flanking sequence corresponding to positions 1 through 10of SEQ ID NO: 4 ([D], see FIG. 2).SEQ ID NO: 3—The 5′ sequence flanking the inserted DNA of MON87705 up tothe T-DNA insertion.SEQ ID NO: 4—The 3′ sequence flanking the inserted DNA of MON87705 up tothe T-DNA insertion.SEQ ID NO: 5—The sequence of the inserted T-DNAs, including residualborder sequence after integration.SEQ ID NO: 6—A 13188 bp nucleotide sequence representing the contig ofthe 5′ sequence flanking the inserted DNA of MON87705 (SEQ ID NO: 3),the sequence of the integrated expression cassette (SEQ ID NO: 5) andthe 3′ sequence flanking the inserted DNA of MON87705 (SEQ ID NO: 4).SEQ ID NO: 7—A 6583 bp nucleotide sequence representing the aroA-CP4expression cassette and the FAD2-1A/FATB suppression cassette asassembled in MON87705 from the co-integration of the pMON95829 T-DNAs.SEQ ID NO: 8—Primer SQ20129 used to identify the MON87705 event. PrimerSQ20129 corresponds to a region in the 3′ of the inserted T-DNA close tothe 3′ left border corresponding to positions 10645 to 10663 of SEQ IDNO: 6. A PCR amplicon produced using the combination of primers SQ20129and SQ20130 is positive for the presence of the event MON87705.SEQ ID NO: 9—Primer SQ20130 used to identify the MON87705. PrimerSQ20130 is complimentary to the region 3′ of the T-DNA insertion bordercorresponding to positions 10688 to 10707 of SEQ ID NO: 6. A PCRamplicon produced using the combination of primers SQ20129 and SQ20130is positive for the presence of the event MON87705.SEQ ID NO: 10—Probe PB10043 used to identify the MON87705 event. Thisprobe is a 6FAM™-labeled synthetic oligonucleotide whose sequencecorresponds to positions 10666 to 10686 of SEQ ID NO: 6. Release of afluorescent signal in an amplification reaction using primers SQ20129and SQ20130 in combination with 6FAM™-labeled probe PB10043 isdiagnostic of event MON87705.SEQ ID NO: 11—Primer SQ21928 used to identify the MON87705 event. PrimerSQ21928 is complimentary to the region 3′ of the T-DNA insertion bordercorresponding to positions 10675 to 10699 of SEQ ID NO: 6. A PCRamplicon produced using the combination of primers SQ20901 and SQ21928is positive for the presence of the event MON87705. Primer SQ21928 isalso used to determine zygosity of MON87705 events. Detection of a PCRamplicon and the release of fluorescent signal using 6FAM™-labeled ProbePB10335 and primers SQ21928 and SQ21905 is positive for presence of wildtype in a zygosity assay. Eighteen base pairs of the sequencecorresponds to positions 1053 to 1070 of SEQ ID NO: 6 with the remainingseven base pairs corresponding to seven base pairs in the forty basepair region of wild type sequence shown as SEQ ID NO: 17 which was lostin the generation of the MON87705 event.SEQ ID NO: 12—Primer SQ20901 used to identify the MON87705 event. PrimerSQ20901 corresponds to a region in the 3′ of the inserted T-DNA close tothe 3′ left border corresponding to positions 10624 to 10647 of SEQ IDNO: 6. A PCR amplicon produced from the combination of primers SQ21928and SQ20901 is positive for the presence of the event MON87705.SEQ ID NO: 13—Probe PB10164 used to identify the MON87705 event. Thisprobe is a 6FAM™-labeled synthetic oligonucleotide whose sequencecorresponds to positions 10651 to 10670 of SEQ ID NO: 6. Release of afluorescent signal in an amplification reaction using primers SQ21928and SQ20901 in combination with 6FAM™-labeled probe PB10164 isdiagnostic of event MON87705.SEQ ID NO: 14—Probe PB10335 used to determine zygosity of MON87705events. This probe is a VIC™-labeled synthetic oligonucleotide whosesequence corresponds to a region of the wild-type genomic DNA. A PCRamplicon produced using primers SQ21928 and SQ21905 causes the releaseof a fluorescent signal using probe PB10335 which is positive for thepresence of the wild-type allele in a zygosity assay for event MON87705.This sequence corresponds to positions 12 to 31 of the forty base pairwild type sequence listed as SEQ ID NO: 17 which was lost in thegeneration of the MON87705 event.SEQ ID NO: 15—Primer SQ21905 used to determine zygosity of MON87705events. Detection of a PCR amplicon using 6FAM™-labeled Probe PB10335and primers SQ21928 and SQ21905 is positive for presence of wild type ina zygosity assay. Sixteen base pairs of sequence corresponds topositions 1037 to 1052 of SEQ ID NO: 6 while this primer continues tenbase pairs into the forty base pair region of wild type sequence listedas SEQ ID NO: 16 which was lost in the generation of the MON87705 event.SEQ ID NO: 16—Forty base pair sequence that was lost at the junction ofthe tandem repeat sequence (SEQ ID NO: 17) and the 5′ genomic sequences.This sequence is found in the wild type and would have been locatedbetween base pair 1052 and 1053 in SEQ ID NO: 6 had it not been lostduring the generation of the MON87705 event. Due to the duplication ofthe genomic sequence upon the generation of the MON87705 event, theterminal twelve bases of sequence is also located at position 10670 to10681 of SEQ ID NO: 6, but the remaining twenty eight base pairs arecompletely absent from the MON87705 event and therefore may be utilizedto identify the wild type in an assay for zygosity.SEQ ID NO: 17—This 2370 bp sequence is the portion of the wild typegenomic sequences which was apparently duplicated upon generation of theMON87705 event. This region of duplication corresponds to position 1053to 3422 and is nearly identical to position 10682 to 13051 of SEQ ID NO:6. Duplicate Genomewalker™ based clones of this region shared twomismatches in the repeat located on the 5′ end of the insert whencompared to the 3′ end and the soy genomic sequence. The two alteredbases in the 5′ repeat, along with the deletion found on the 5′ end ofthe duplication (SEQ ID NO:16) leads us to the additional that thetandem sequences corresponding to position 1053 to 3422 is the newlygenerated sequence while the sequence corresponding to position 10682 to13051 is the original wild type sequence.SEQ ID NO: 18—This 106 bp sequence spans the right border to rightborder junction generated upon the co-integration of the two pMON95829T-DNAs to yield event MON87705. It starts and ends with twenty bases ofthe suppression target FATB, one from each arm of the inverted repeat,and contains residual border sequences and inserted sequences generatedin the integration of the sequences found in the MON87705 event. Thissequence corresponds to position 9230 to 9335 of SEQ ID NO: 6. Althoughthis sequence lies within the suppression cassette, due to the randomnature of the assembly of the two T-DNAs in vivo, the sequencecombination is unique to the MON87705 event.SEQ ID NO: 19—Primer 24894 used in the secondary (nested) PCR incombination with AP2 for amplification of the Genomewalker™-derived 5′flank extension. This sequence corresponds to position 3722 to 3748 ofSEQ ID NO: 6.SEQ ID NO: 20—Primer 24895 used in the primary PCR in combination withAP1 for amplification of the Genomewalker™-derived 5′ flank extension.This sequence corresponds to position 3798 to 3827 of SEQ ID NO: 6.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art. Definitions of common terms in molecular biologymay also be found in Rieger et al., Glossary of Genetics: Classical andMolecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin,Genes V, Oxford University Press: New York, 1994.

As used herein, the term “soybean” means Glyycine max and includes allplant varieties that can be bred with soybean, including wild soybeanspecies as well as those plants belonging to Glycine soja that permitbreeding between species.

“Glyphosate” refers to N-phosphonomethylglycine and its salts.N-phosphonomethylglycine is a well-known herbicide that has activity ona broad spectrum of plant species.

“Desaturase” refers to a polypeptide that can desaturate or catalyzeformation of a double bond between consecutive carbons of one or morefatty acids to produce a mono- or poly-unsaturated fatty acid or aprecursor thereof. Of particular interest are polypeptides that cancatalyze the conversion of oleic acid to linoleic acid or linoleic acidto α-linolenic acid, which includes enzymes which desaturate at the 12or 15 positions. Considerations for choosing a specific polypeptidehaving desaturase activity include, but are not limited to, the pHoptimum of the polypeptide, whether the polypeptide is a rate limitingenzyme or a component thereof, whether the desaturase used is essentialfor synthesis of a desired PUFA, and/or whether a co-factor is requiredby the polypeptide. The expressed polypeptide preferably hascharacteristics that are compatible with the biochemical environment ofits location in the host cell. For example, the polypeptide may have tocompete for substrate(s). An endogenous desaturase may also be thetarget of gene suppression.

“Thioesterase” refers to a polypeptide that can hydrolyze the thioesterbond of molecules (splitting of an ester bond into acid and alcohol, inthe presence of water) specifically at a thiol group. Of particularinterest are polypeptides that can catalyze the hydrolysis of thethioester bond contained in acyl-acyl-carrier proteins (acyl-ACP),especially stearoyl- and palmitoyl-ACP substrates. Such hydrolysisproduces a free fatty acid and ACP, thereby terminating the fatty acidbiosynthesis of plants located in the plastid and readies the fatty acidfor export to the cytoplasm. Considerations for choosing a specificpolypeptide having thioesterase activity include, but are not limitedto, the pH optimum of the polypeptide, whether the polypeptide is a ratelimiting enzyme or a component thereof and whether the thioesterase usedis essential for increased production of saturated fatty acids. Theexpressed polypeptide preferably has characteristics that are compatiblewith the biochemical environment of its location in the host cell. Forexample, the polypeptide may have to compete for substrate(s). Anendogenous thioesterase may also be the target of gene suppression.

A “commodity product” refers to any product which is comprised ofmaterial derived from soybean or soybean oil and is sold to consumers.Processed soybeans are the largest source of protein feed and vegetableoil in the world. The soybean plant MON87705 can be used to manufacturecommodities typically acquired from soy. Soybeans of MON87705 can beprocessed into meal, flour, or oil as well as be used as a protein oroil source in animal feeds for both terrestrial and aquatic animals.Soybeans and soybean oils from MON87705 can be used in the manufactureof many different products, not limited to, nontoxic plastics, printinginks, lubricants, waxes, hydraulic fluids, electric transformer fluids,solvents, cosmetics, and hair care products. Soybeans and oils ofMON87705 can be suitable for use in a variety of soyfoods made fromwhole soybeans, such as soymilk, soy nut butter, natto, and tempeh, andsoyfoods made from processed soybeans and soybean oil, including soybeanmeal, soy flour, soy protein concentrate, soy protein isolates,texturized soy protein concentrate, hydrolyzed soy protein, whippedtopping, cooking oil, salad oil, shortening, and lecithin. Wholesoybeans are also edible, and are typically sold to consumers raw,roasted, or as edamamé. Soymilk, which is typically produced by soakingand grinding whole soybeans, may be consumed without other processing,spray-dried, or processed to form soy yogurt, soy cheese, tofu, or yuba.

Oils of MON87705 can be used to make biodiesel. The use of biodiesel inconventional diesel engines results in substantial reductions ofpollutants such as sulfates, carbon monoxide, and particulates comparedto petroleum diesel fuel, and use in school buses can greatly reduceexposure to toxic diesel exhaust. Biodiesel is typically obtained byextracting, filtering and refining soybean oil to remove free fats andphospholipids, and then transesterifying the oil with methanol to formmethyl esters of the fatty acids (see for example U.S. Pat. No.5,891,203). The resultant soy methyl esters are commonly referred to as“biodiesel.” The oil derived from MON87705 may also be used as a dieselfuel without the formation of methyl esters, such as, for example, bymixing acetals with the oil (see for example U.S. Pat. No. 6,013,114).The seeds of MON87705 used to make said oils can be identified by themethods of the present invention. It is expected that purified oil fromMON87705 event seeds or mixtures of seeds some or all of which areMON87705 will have relatively no DNA available for testing. However, theseeds from which the oils are extracted can be characterized with themethod of the present invention to identify the presence of the MON87705event within the population of seeds used to make said oils. Also, plantwaste from the process used to make said oils can be used in the methodsof the present invention to identify the presence of MON87705 eventswithin a mixture of seeds processed to make said oils. Likewise, plantdebris left after making a commodity product, or left behind followingharvest of the soybean seed, can be characterized by the methods of thepresent invention to identify MON87705 events within the raw materialsused to make said commodity products.

The present invention also includes a blended or non-blended soybean oilof MON87705. Such oil may be blended with other oils. In a preferredembodiment, the oil produced from plants of the present invention orgenerated by a method of the present invention constitutes greater than0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weight of the oilcomponent of any product. In another embodiment, the oil preparation maybe blended and can constitute greater than 10%, 25%, 35%, 50% or 75% ofthe blend by volume. Oil produced from a plant of the present inventioncan be admixed with one or more organic solvents or petroleumdistillates.

A transgenic “event” is produced by transformation of plant cells withheterologous DNA, i.e., a nucleic acid construct that includes atransgene of interest, regeneration of a population of plants resultingfrom the insertion of the transgene into the genome of the plant, andselection of a particular plant characterized by insertion into aparticular genome location. The term “event” refers to the originaltransformant and progeny of the transformant that include theheterologous DNA. The term “event” also refers to progeny produced by asexual outcross between the transformant and another variety, whereinthe progeny comprises the heterologous DNA. Even after repeatedback-crossing to a recurrent parent, the inserted DNA and flanking DNAfrom the original transformation may be present in the progeny of thecross at the same chromosomal location. The term “event” also refers toDNA from the original transformant comprising the inserted DNA andflanking genomic sequence immediately adjacent to the inserted DNA thatwould be expected to be transferred to a progeny that receives insertedDNA including the transgene of interest as the result of a sexual crossof one parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA. The present invention relates tothe event MON87705 DNA, plant cells, tissues, seeds and processedproducts derived from MON87705.

It is also to be understood that two different transgenic plants, or atransgenic plant and a wild-type plant, can also be mated to produceoffspring that contain two independently segregating added, exogenousgenes. The offspring can be homozygous or heterozygous for the genes.Selfing of appropriate progeny can produce plants that are homozygousfor both added, exogenous genes. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation. Descriptions of other breeding methods that arecommonly used for different traits and crops can be found in one ofseveral references, e.g., Fehr, in Breeding Methods for CultivarDevelopment, Wilcox J. ed., American Society of Agronomy, Madison Wis.(1987).

As used herein when referring to an “isolated DNA molecule”, it isintended that the DNA molecule be one that is present, alone or incombination with other compositions, but not within its naturalenvironment. For example, a coding sequence, intron sequence,untranslated leader sequence, promoter sequence, transcriptionaltermination sequence, and the like, that are naturally found within theDNA of a soybean genome are not considered to be isolated from thesoybean genome so long as they are within the soybean genome. However,each of these components, and subparts of these components, would be“isolated” within the scope of this disclosure so long as the structuresand components are not within the soybean genome. For the purposes ofthis disclosure, any transgenic nucleotide sequence, i.e., thenucleotide sequence of the DNA inserted into the genome of the cells ofthe soybean plant event MON87705 would be considered to be an isolatednucleotide sequence whether it is present within the plasmid used totransform soybean cells from which the MON87705 event arose, within thegenome of the event MON87705, present in detectable amounts in tissues,progeny, biological samples or commodity products derived from the eventMON87705. The nucleotide sequence or any fragment derived therefromwould therefore be considered to be isolated or isolatable if the DNAmolecule can be extracted from cells, or tissues, or homogenate from aplant or seed or plant organ; or can be produced as an amplicon fromextracted DNA or RNA from cells, or tissues, or homogenate from a plantor seed or plant organ, any of which is derived from such materialsderived from the event MON87705. For that matter, the junction sequencesas set forth at SEQ ID NOs: 1, 2 and 18, and nucleotide sequencesderived from event MON87705 that also contain these junction sequencesare considered to be isolated or isolatable, whether these sequences arepresent within the genome of the cells of event MON87705 or present indetectable amounts in tissues, progeny, biological samples or commodityproducts derived from the event MON87705.

A DNA molecule of the present invention may also be a recombinant DNAmolecule. As used herein, the term recombinant means any agent (e.g.DNA, peptide etc.), that is, or results, however indirect, from humanmanipulation of a nucleic acid molecule.

A “probe” is an isolated nucleic acid to which is attached aconventional detectable label or reporter molecule, e.g., a radioactiveisotope, ligand, chemiluminescent agent, or enzyme. Such a probe iscomplementary to a strand of a target nucleic acid, in the case of thepresent invention, to a strand of genomic DNA from soybean eventMON87705 whether from a soybean plant or from a sample that includes DNAfrom the event. Probes according to the present invention include notonly deoxyribonucleic or ribonucleic acids but also polyamides and otherprobe materials that bind specifically to a target DNA sequence and suchbinding can be used to detect the presence of that target DNA sequence.

“Primers” are isolated nucleic acids that are annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a polymerase, e.g., a DNA polymerase.Primer pairs of the present invention refer to their use foramplification of a target nucleic acid sequence, e.g., by the polymerasechain reaction (PCR) or other conventional nucleic-acid amplificationmethods.

Probes and primers are generally 11 nucleotides or more in length,preferably 15 nucleotides or more, more preferably 18 nucleotides ormore, more preferably 20 nucleotides or more, more preferably 24nucleotides or more, and most preferably 30 nucleotides or more. Suchprobes and primers hybridize specifically to a target sequence underhigh stringency hybridization conditions. Preferably, probes and primersaccording to the present invention have complete sequence similaritywith the target sequence, although probes differing from the targetsequence and that retain the ability to hybridize to target sequencesmay be designed by conventional methods.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989 (hereinafter, “Sambrook et al., 1989”); CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates)(hereinafter, “Ausubel et al., 1992”); and Innis et al., PCR Protocols:A Guide to Methods and Applications, Academic Press: San Diego, 1990.PCR-primer pairs can be derived from a known sequence, for example, byusing computer programs intended for that purpose such as Primer(Version 0.5, © 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences.

The nucleic acid probes and primers of the present invention hybridizeunder stringent conditions to a target DNA sequence. Any conventionalnucleic acid hybridization or amplification method can be used toidentify the presence of DNA from a transgenic event in a sample.Nucleic acid molecules or fragments thereof are capable of specificallyhybridizing to other nucleic acid molecules under certain circumstances.As used herein, two nucleic acid molecules are said to be capable ofspecifically hybridizing to one another if the two molecules are capableof forming an anti-parallel, double-stranded nucleic acid structure. Anucleic acid molecule is said to be the “complement” of another nucleicacid molecule if they exhibit complete complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the molecules is complementary to a nucleotide ofthe other. Two molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook et al., 1989, and by Haymes et al.,In: Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985). Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. In order for a nucleic acid molecule to serve as a primer orprobe it need only be sufficiently complementary in sequence to be ableto form a stable double-stranded structure under the particular solventand salt concentrations employed.

As used herein, a substantially homologous sequence is a nucleic acidsequence that will specifically hybridize to the complement of thenucleic acid sequence to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6.0× sodium chloride/sodium citrate(SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C., areknown to those skilled in the art or can be found in Current Protocolsin Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. In aembodiment, a nucleic acid of the present invention will specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 1 and 2 or complements thereof or fragments of either undermoderately stringent conditions, for example at about 2.0×SSC and about65° C. In a particularly preferred embodiment, a nucleic acid of thepresent invention will specifically hybridize to one or more of thenucleic acid molecules set forth in SEQ ID NO:1 and SEQ ID NO: 2 orcomplements or fragments of either under high stringency conditions. Inone aspect of the present invention, a marker nucleic acid molecule ofthe present invention has the nucleic acid sequence set forth in SEQ IDNO: 1 and SEQ ID NO: 2 or complements thereof or fragments of either. Inanother aspect of the present invention, a marker nucleic acid moleculeof the present invention shares 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 912%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%sequence identity with the nucleic acid sequence set forth in SEQ ID NO:1 and SEQ ID NO: 2 or complement thereof or fragments of either. In afurther aspect of the present invention, a marker nucleic acid moleculeof the present invention shares 95% 96%, 97%, 98%, 99% and 100% sequenceidentity with the sequence set forth in SEQ ID NO: 1 and SEQ ID NO: 2 orcomplement thereof or fragments of either. SEQ ID NO: 1 and SEQ ID NO: 2may be used as markers in plant breeding methods to identify the progenyof genetic crosses similar to the methods described for simple sequencerepeat DNA marker analysis, in “DNA markers: Protocols, applications,and overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY; allof which is herein incorporated by reference. The hybridization of theprobe to the target DNA molecule can be detected by any number ofmethods known to those skilled in the art, these can include, but arenot limited to, fluorescent tags, radioactive tags, antibody based tags,and chemiluminescent tags.

Regarding the amplification of a target nucleic acid sequence (e.g., byPCR) using a particular amplification primer pair, “stringentconditions” are conditions that permit the primer pair to hybridize onlyto the target nucleic-acid sequence to which a primer having thecorresponding wild-type sequence (or its complement) would bind andpreferably to produce a unique amplification product, the amplicon, in aDNA thermal amplification reaction.

The term “specific for (a target sequence)” indicates that a probe orprimer hybridizes under stringent hybridization conditions only to thetarget sequence in a sample comprising the target sequence.

As used herein, “amplified DNA” or “amplicon” refers to the product ofnucleic-acid amplification of a target nucleic acid sequence that ispart of a nucleic acid template. For example, to determine whether thesoybean plant resulting from a sexual cross contains transgenic eventgenomic DNA from the soybean plant of the present invention, DNAextracted from a soybean plant tissue sample may be subjected to nucleicacid amplification method using a primer pair that includes a primerderived from flanking sequence in the genome of the plant adjacent tothe insertion site of inserted heterologous DNA, and a second primerderived from the inserted heterologous DNA to produce an amplicon thatis diagnostic for the presence of the event DNA. The amplicon is of alength and has a sequence that is also diagnostic for the event. Theamplicon may range in length from the combined length of the primerpairs plus one nucleotide base pair, preferably plus about fiftynucleotide base pairs, more preferably plus about two hundred-fiftynucleotide base pairs, and even more preferably plus about fourhundred-fifty nucleotide base pairs. Alternatively, a primer pair can bederived from flanking sequence on both sides of the inserted DNA so asto produce an amplicon that includes the entire insert nucleotidesequence. A member of a primer pair derived from the plant genomicsequence may be located a distance from the inserted DNA molecule, thisdistance can range from one nucleotide base pair up to about twentythousand nucleotide base pairs. The use of the term “amplicon”specifically excludes primer-dimers that may be formed in the DNAthermal amplification reaction.

Nucleic-acid amplification can be accomplished by any of the variousnucleic-acid amplification methods known in the art, including thepolymerase chain reaction (PCR). A variety of amplification methods areknown in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and in PCR Protocols: A Guide to Methods andApplications, ed. Innis et al., Academic Press, San Diego, 1990. PCRamplification methods have been developed to amplify up to 22 kb ofgenomic DNA and up to 42 kb of bacteriophage DNA (Cheng et al., Proc.Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as othermethods known in the art of DNA amplification may be used in thepractice of the present invention. The sequence of the heterologous DNAinsert or flanking sequence from soybean event MON87705 with seedsamples deposited as ATCC PTA-9241 can be verified (and corrected ifnecessary) by amplifying such sequences from the event using primersderived from the sequences provided herein followed by standard DNAsequencing of the PCR amplicon or of the cloned DNA.

The amplicon produced by these methods may be detected by a plurality oftechniques. One such method is Genetic Bit Analysis (Nikiforov, et al.Nucleic Acid Res. 22:4167-4175, 1994) where an DNA oligonucleotide isdesigned which overlaps both the adjacent flanking genomic DNA sequenceand the inserted DNA sequence. The oligonucleotide is immobilized inwells of a microwell plate. Following PCR of the region of interest(using one primer in the inserted sequence and one in the adjacentflanking genomic sequence), a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labelledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another method is the Pyrosequencing technique as described by Winge(Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotideis designed that overlaps the adjacent genomic DNA and insert DNAjunction. The oligonucleotide is hybridized to single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking genomic sequence) and incubated in the presenceof a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., (Genome Res.9:492-498, 1999) is a method that can be used to detect the amplicon ofthe present invention. Using this method an oligonucleotide is designedwhich overlaps the genomic flanking and inserted DNA junction. Theoligonucleotide is hybridized to single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking genomic DNA sequence) and incubated in the presence of a DNApolymerase and a fluorescent-labeled ddNTP. Single base extensionresults in incorporation of the ddNTP. Incorporation can be measured asa change in polarization using a fluorometer. A change in polarizationindicates the presence of the transgene insert/flanking sequence due tosuccessful amplification, hybridization, and single base extension.

TaqMan® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps thegenomic flanking and insert DNA junction. The FRET probe and PCR primers(one primer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al. (Nature Biotech. 14:303-308, 1996) Briefly,a FRET oligonucleotide probe is designed that overlaps the flankinggenomic and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties that results in the production of afluorescent signal. The fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Other described methods, such as microfluidics (US Patent Pub.2006068398, U.S. Pat. No. 6,544,734) provide methods and devices toseparate and amplify DNA samples. Optical dyes are used to detect andquantitate specific DNA molecules (WO/05017181). Nanotube devices(WO/06024023) that comprise an electronic sensor for the detection ofDNA molecules or nanobeads that bind specific DNA molecules and can thenbe detected.

DNA detection kits can be developed using the compositions disclosedherein and the methods well known in the art of DNA detection. The kitsare useful for the identification of soybean event MON87705 DNA in asample and can be applied to methods for breeding soybean plantscontaining the appropriate event DNA. The kits may contain DNA primersor probes that are homologous or complementary to SEQ ID NO: 1 throughSEQ ID NO: 5 or DNA primers or probes homologous or complementary to DNAcontained in the transgene genetic elements of DNA. These DNA sequencescan be used in DNA amplification reactions or as probes in a DNAhybridization method. The sequences of the genomic DNA and transgenegenetic elements contained in MON87705 soybean genome are illustrated inFIG. 2; the transgene genetic element contained in the T-DNAs areorganized as follows: the first T-DNA begins with the octopine leftborder sequence, followed by the first artificial gene comprised of the35S enhancer from figwort mosaic virus (FMV), and promoter, intron, andleader sequence from the Arabidopsis thaliana Tsf1 gene, which isupstream of the ShkG transit peptide fused to an optimized aroA-CP4,which is upstream of the 3′ UTR of the RbcS2 (E9) gene from Pisumsativum, followed by the second cassette which is comprised of thepromoter and leader sequence from the Glycine max 7S alpha prime subunitof beta-conglycinin gene, which is upstream of the sense half of theinverted repeat containing sequences homologous to the Glycine max FAD2and FATB genes, which is upstream of the nopaline right border sequence.The second T-DNA begins with the nopaline right border sequence, whichis upstream of the antisense half of the inverted repeat containingsequences homologous to the Glycine max FAD2 and FATB genes, which isupstream of the 3′ UTR of the Gossypium barbadense (Sea island cotton)H6 gene, which is upstream of the octopine left border sequence. Theprimer molecules derived from these sequences can be used as part of aprimer set that also includes a DNA primer molecule derived from thegenome flanking the transgene insert of event MON87705 as presented inSEQ ID NO: 3 and SEQ ID NO: 4.

The present invention includes a soybean plant comprising a DNA moleculecomprising a polynucleotide having a sequence that is or iscomplementary to a sequence selected from the group consisting of SEQ IDNOs: 1, 2 and 18. In another aspect, the present invention includes asoybean plant part, where the plant part comprises a polynucleotidehaving a sequence that is or is complementary to a sequence selectedfrom the group consisting of SEQ ID NOs: 1, 2 and 18. In another aspect,the present invention includes progeny of a soybean plant, where theprogeny comprises a polynucleotide having a sequence that is or iscomplementary to a sequence selected from the group consisting of SEQ IDNOs: 1, 2 and 18.

The present invention also includes a soybean plant capable of producingseeds having an oil composition comprising approx. 55-80% oleic acid andless than 8% saturated fatty acid, wherein genetic determinants for saidoil composition is obtainable from soybean having ATCC Accession No.PTA-9241. In another aspect, the present invention includes a method forproducing a soybean plant comprising altered fatty acid levels,comprising crossing a plant comprising soybean event MON87705 with asoybean plant lacking soybean event MON87705 to obtain a plantcomprising said soybean event MON87705 and altered fatty acid levels,wherein a representative sample of seed comprising said event wasdeposited under ATCC Accession No. PTA-9241. In a further aspect, thepresent invention also includes a method of producing a soybean varietycomprising soybean event MON87705, comprising backcrossing soybean eventMON87769 into said variety, wherein a representative sample of seedcomprising said event was deposited under ATCC Accession No. PTA-9241.

The present invention also includes an oil composition obtained fromseeds comprising a DNA molecule comprising a polynucleotide having asequence that is or is complementary to a sequence selected from thegroup consisting of SEQ ID NOs: 1, 2 and 18. In another aspect, thepresent invention includes a commodity product derived from an oilcomposition selected from the group consisting of cooking oil, saladoil, shortening, lecithin, nontoxic plastics, printing inks, lubricants,waxes, hydraulic fluids, electric transformer fluids, solvents,cosmetics, hair care products and biodiesel.

The present invention also includes a DNA molecule comprising apolynucleotide having a sequence that is or is complementary to oneselected from the group consisting of sequences listed under SEQ ID NOs:1, 2 and 18. In another aspect, the present invention includes anisolated DNA molecule comprising at least from about 11 to about 20consecutive nucleotides selected from the group consisting of SEQ ID NO:1 and SEQ ID NO: 2.

The present invention also includes a genome of a soybean cellcomprising a polynucleotide having a sequence selected from the groupconsisting of sequences listed under SEQ ID NOs: 1, 2 and 18.

In another aspect, the present invention includes a method of detectingthe presence of soybean event MON87705 DNA in a biological samplecomprising contacting the sample with a probe that hybridizes understringent hybridization conditions with a sequence selected from thegroup consisting of SEQ ID NO: 1 and SEQ ID NO: 2, and theircomplements, and does not hybridize under stringent hybridizationconditions with soybean plant genomic DNA that that does not comprise asequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2, and their complements; subjecting the sample and probe tostringent hybridization conditions; and detecting binding of the probeto the sample; where binding is diagnostic for the presence of said DNAin the sample.

In another aspect, the present invention includes a method for detectingthe presence of a nucleotide sequence diagnostic for the presence ofsoybean event MON87705 in a biological sample, comprising detecting thepresence of a nucleotide sequence wherein said sequence is selected fromthe group consisting of SEQ ID NOs: 1, 2 and 18, wherein said biologicalsample is selected from the group consisting of soybean meal, soy flour,soy protein concentrate, soy protein isolates, texturized soy proteinconcentrate, hydrolyzed soy protein and whipped topping.

The present invention also includes a kit for detecting the presence orabsence of soybean transgenic event MON87705 in a soybean samplecomprising nucleotide components designed based on detecting one or moresequences shown under SEQ ID NOs: 1, 2 and 18. In another aspect, thepresent invention also includes a composition having a DNA moleculeselected from the group consisting of SEQ ID NOs: 1, 2 and 18, whereinsaid composition is a commodity product selected from the groupconsisting of soybean meal, soy flour, soy protein concentrate, soyprotein isolates, texturized soy protein concentrate, hydrolyzed soyprotein and whipped topping.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Transformation of Soybean A3525 with pMON95829 andEvent Selection

The transgenic soybean plant MON87705 was generated by anAgrobacterium-mediated transformation of soybean cells with DNAfragments derived from pMON95829 (FIG. 1). The binary planttransformation vector, pMON95829 contains two plant transformationT-DNAs. Each T-DNA was flanked by right border (RB) and left border (LB)sequences at the ends of the T-DNAs. Post transformation screening ofMON87705 identified a right border to right border co-integration of thetwo T-DNAs generating a two cassette insertion, one designed to expressthe aroA-CP4 gene from Agrobacterium tumefaciens imparting glyphosatetolerance and the other designed with an inverted repeat to trigger theRNAi based suppression of the endogenous FAD2 and FATB genes (SEQ ID NO:7). The inverted repeat structure is not found in pMON95829, but isformed with a RB to RB co-integration of the two T-DNAs. Thisintegration configuration yields a single transgenic locus containingboth expression and suppression cassettes and is flanked by residualleft border sequences (FIG. 2). The unique sequence generated at theRB:RB integration site is listed under SEQ ID NO: 18.

The T-DNAs are organized as follows: the first T-DNA begins with theoctopine LB sequence, followed by the first artificial gene comprised ofthe 35S enhancer from figwort mosaic virus (FMV) and the promoter,intron, and leader sequence from the Arabidopsis thaliana Tsf1 gene,which is upstream of the ShkG transit peptide fused to an optimizedaroA-CP4, which is upstream of the 3′ UTR of the RbcS2 (E9) gene fromPisum sativum, followed by the second cassette which is comprised of thepromoter and leader sequence from the Glycine max 7S alpha prime subunitof beta-conglycinin gene, which is upstream of the sense half of theinverted repeat containing sequences homologous to the Glycine max FAD2and FATB genes, which is upstream of the nopaline RB sequence. Thesecond T-DNA begins with the nopaline RB sequence, which is upstream ofthe antisense half of the inverted repeat containing sequenceshomologous to the Glycine max FAD2 and FATB genes, which is upstream ofthe 3′ UTR of the Gossypium barbadense (Sea island cotton) H6 gene,which is upstream of the octopine LB sequence.

Explants transformed with pMON95829 were obtained via Agrobacteriumtumefaciens-mediated transformation. Plants were regenerated fromtransformed tissue. Approximately 955 R0 transformation events wereproduced and were tested for the presence of the two T-DNAs byInvader^(R) (Third Wave Technologies, Inc., Madison, Wis.). In addition,a PCR designed to identify the RB:RB configuration was used to selectproperly-assembled candidate events. R0 events demonstrating the properco-integration of the T-DNAs were self-pollinated to generate R1 seed.The fatty acid composition of the R1 seed was determined by FAME-GCanalysis. Based on these analyses, 48 events were carried forward to theR2 generation. Segregation of multi copy events and phenotypiccharacterization narrow the candidate event pool to 11 events by the R3generation.

Detailed southern analysis designed to verify copy number, cassetteintactness, locus and absence of undesired vector sequence wasperformed. In subsequent generations, field performance characteristics,as well as sequence flanking the insertion site for the remainingevents, were also determined. One progeny line designated event MON87705was selected based upon its overall fatty acid composition profile(Table 1), agronomic performance and molecular characteristics.

TABLE 1 Fatty Acid Composition Profile (weight % of total fatty acids)Soy Line 16:0 18:0 18:1 18:2 18:3 Wild type 12.2 4.2 19.0 54.9 8.0MON87705 2.5 3.4 72.8 12.3 7.4

Example 2 Isolation of Flanking Sequences Using Inverse PCR

Sequences flanking the T-DNA insertion in MON87705 were determined usinginverse PCR as described in Ochman et al., 1990 (PCR Protocols: A guideto Methods and Applications, Academic Press, Inc.) and by TAIL (ThermalAsymmetric InterLaced) PCR. Plant genomic DNA was isolated from bothwild-type A3525 and the transgenic line from tissue grown under greenhouse conditions. Frozen leaf tissue was ground by mortar and pestlewith liquid nitrogen or mechanical grinding. A volume of 22 mL ofextraction buffer was added to ˜1 g of ground leaf tissue and incubatedat 65° C. for 1 hour. The CTAB extraction buffer consisted of 1.4M NaCl,2% CTAB, 20 mM EDTA, and 100 mM Tris-HCl pH 8.0. Just prior to use,0.02% beta-mercaptoethanol and 0.5 mg RNase A was added to theextraction buffer. The samples were extracted with 12 mL ofphenol/chloroform/isoamyl alcohol (25:24:1) solution and thencentrifuged at 4000×G for 10 minutes at 4° C. The supernatant wastransferred to a new tube and the DNA was precipitated with 15 mL ofisopropanol. After centrifugation at 4000×G for 10 minutes, the pelletswere washed with 5 mL 70% ethanol. A final centrifugation at 4000×G for5 minutes was performed; the pellets were air dried and thenre-suspended in 300 μL of water.

An aliquot of DNA was subjected to TAIL PCR and a region of sequenceadjacent to the insertion site was isolated and sequenced. Additionallyan aliquot of DNA was digested with restriction endonucleases selectedbased upon restriction analysis of the T-DNA. After self-ligation ofrestriction fragments, PCR was performed using primers designed from theT-DNA sequence that would amplify sequences extending away from the 5′and 3′ ends of the T-DNA. PCR products were separated by agarose gelelectrophoresis and purified using a QIAGEN gel purification kit(Qiagen, Valencia, Calif.). The subsequent products were sequenceddirectly using standard sequencing protocols.

Extension of the initial flanking sequence extending away from the 5′end of the T-DNA was performed using the Genomewalker™ kit (Clontech,Mountain View, Calif.). Standard protocol conditions were used andprimers presented as SEQ ID NO: 19 and SEQ ID No: 20 were used in thenested PCR along with the supplied AP1 and AP2 primers. Reactions wererun in duplicate and products were cloned and sequenced directly usingstandard sequencing protocols. Sequences were compared to the duplicaterun, prior genomic sequences, and the 3′ flanking sequence to determineactual polymorphisms from PCR-derived errors. There is apparently achromosomal duplication that occurred upon generation of the MON87705event. This region of duplication (SEQ ID NO: 17) corresponds toposition 1053 through 3422 of SEQ ID NO: 6 and is nearly identical toposition 10682 through 13051 of SEQ ID NO: 6. We have determined thatthere are two polymorphisms between the repeat located on the 5′ end ofthe insert and the repeat from the 3′ end of the insert, with the 3′ endof the insert matching the original genomic sequence.

Using these methods, the identified 5′ flanking sequence of theinsertion was presented as SEQ ID NO: 3 (see FIG. 2), and the 3′flanking sequence was presented as SEQ ID NO: 4 (see FIG. 2). Theportion of the inserted cassettes (SEQ ID NO: 7) from pMON95829 that wasfully integrated into the A3525 genomic DNA is presented as SEQ ID NO: 5(see FIG. 2).

Isolated sequences were compared to the T-DNA sequence to identify theflanking sequence and the co-isolated T-DNA fragment. Confirmation ofthe presence of the expression cassette was achieved by PCR with primersdesigned based upon the deduced flanking sequence data and the knownT-DNA sequence. The A3525 wild type sequence corresponding to the sameregion in which the T-DNA was integrated in the transformed line wasisolated using primers designed from the flanking sequences in MON87705.The flanking sequences in MON87705 and the A3525 wild type sequence wereanalyzed against multiple nucleotide and protein databases. Thisinformation was used to examine the relationship of the transgene to theplant genome and to look at the insertion site integrity. The flankingsequence and wild type sequences were used to design primers for TaqManendpoint assays used to identify the events and determine zygosity asdescribed in Example 3.

Example 3 Event-Specific Endpoint Taqman and Zygosity Assays

The methods used to identify event MON87705 in a sample areevent-specific endpoint TaqMan PCR assays for which examples ofconditions are described in Table 2 and Table 3. The first set of DNAprimers that may be used in the endpoint assays are primers SQ20129 (SEQID NO: 8), SQ20130 (SEQ ID NO: 9) and 6FAM™ labeled primer PB10043 (SEQID NO: 10). The second set of DNA primers that may be used in theendpoint assays are primers SQ21928 (SEQ ID NO:11), SQ20901 (SEQ IDNO:12), and 6FAM™ labeled primer PB10164 (SEQ ID NO:13). 6FAM™ is afluorescent dye product of Applied Biosystems (Foster City, Calif.)attached to the DNA primers. For TaqMan MGB (Minor Groove Binding)probes, the 5′exonuclease activity of Taq DNA polymerase cleaves theprobe from the 5′-end, between the fluorophore and quencher. Whenhybridized to the target DNA strand, quencher and fluorophore areseparated enough to produce a fluorescent signal.

SQ20129 (SEQ ID NO: 8) and SQ20130 (SEQ ID NO: 9) when used as describedwith PB10043 (SEQ ID NO: 10) produce a DNA amplicon that is diagnosticfor event MON87705 DNA. SQ21928 (SEQ ID NO: 11) and SQ20901 (SEQ ID NO:12) when used as described with PB10164 (SEQ ID NO: 13) produce a DNAamplicon that is also diagnostic for event MON87705 DNA. The controlsfor these analyses should include a positive control from soybean knownto contain event MON87705 DNA, a negative control from non-transgenicsoybean and a negative control that contains no template DNA. Similarassays can be designed for SEQ ID 18

These assays are optimized for use with an Applied Biosystems GeneAmpPCR System 9700, Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 orEppendorf Mastercycler Gradient thermocycler. Other methods andapparatus may be known to those skilled in the art to produce ampliconsthat identify the event MON87705 DNA.

DNA amplification in a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, Eppendorf Mastercycler Gradient thermocycler, Applied BiosystemsGeneAmp PCR System 9700 or MJ Research DNA Engine PTC-225 thermal cycleris performed using the following cycling parameters. When running thePCR in the Eppendorf Mastercycler Gradient or MJ Engine, thethermocycler should be run in the calculated mode. When running the PCRin the Perkin-Elmer 9700, the thermocycler is run with the ramp speedset at maximum.

TABLE 2 Soybean MON87705 Event Specific Endpoint TaqMan PCR Assay StepReagent Volume Comments 1 18 megohm water adjust for final volume of 10μl 2 2X Universal Master Mix   5 μl 1X final concentration of buffer 3Event Primer-SQ20129 and SQ20130 Mix 0.5 μl 1.0 μM final (or eventprimer SQ21928 and SQ20901) concentration (resuspended in 18 megohmwater to a concentration of 20 μM for each primer) Example: In amicrocentrifuge tube, the following should be added to achieve 500 μl ata final concentration of 20 μM: 100 μl of Primer 1 at a concentration of100 μM 100 μl of Primer 2 at a concentration of 100 μM 300 μl of 18megohm water 4 Event 6-FAM ™ Probe PB10043 or 0.2 μl 0.2 μM finalPB10164 concentration (resuspended in 18 megohm water to a concentrationof 10 μM) Note: 6-FAM ™ Probe is light sensitive. 5 Internal ControlPrimer-SQ1532, SQ1533 0.5 μl 1.0 μM final Mix (resuspended in 18 megohmwater to a concentration concentration of 20 μM for each primer) 6Internal Control VIC ™ Probe PB359 0.2 μl 0.2 μM final (resuspended in18 megohm water to a concentration concentration of 10 μM Note: VIC ™Probe is light sensitive. 7 Extracted DNA (template): 3.0 μl LeafSamples to be analyzed Negative control (non-transgenic DNA) Negativewater control (No template control) Positive control GM_A94978 DNA

TABLE 3 Endpoint TaqMan thermocycler conditions Cycle No. Settings 1 50°C. 2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64° C. 1 minute−1° C./cycle 30 95° C. 15 seconds 54° C. 1 minute 1 10° C. Forever

Determining zygosity for event MON87705 in a sample was done using anevent-specific zygosity endpoint TaqMan PCR assay for which examples ofconditions are described in Table 4 and Table 5. The DNA primers used inthe zygosity assay are primers SQ20901 (SEQ ID NO: 12), SQ21928 (SEQ IDNO: 11), SQ21905 (SEQ ID NO: 15), 6FAM™ labeled primer PB10164 (SEQ IDNO: 13) and VIC™ labeled primer PB10335 (SEQ ID NO: 14). 6FAM™ and VIC™are fluorescent dye products of Applied Biosystems (Foster City, Calif.)attached to the DNA primers.

SQ20901 (SEQ ID NO: 12) and SQ21928 (SEQ ID NO: 11) when used in thesereaction methods with PB10164 (SEQ ID NO: 13) produce a DNA ampliconthat is diagnostic for event MON87705 DNA. The controls for thisanalysis should include a positive control from soybean containing eventMON87705 DNA, a negative control from non-transgenic soybean and anegative control that contains no template DNA. SQ21928 (SEQ ID NO: 11)and SQ21905 (SEQ ID NO: 15) when used in these reaction methods withPB10335 (SEQ ID NO: 14) produce a DNA amplicon that is diagnostic forthe wild type allele and contains SEQ ID NO: 16 in its entirety.Heterozygosity is determined by the presence of both ampliconsdemonstrated by the liberation of fluorescent signal from both probesPB10164 and PB10335.

These assays are optimized for use with an Applied Biosystems GeneAmpPCR System 9700, Stratagene Robocycler, MJ Engine, Perkin-Elmer 9700 orEppendorf Mastercycler Gradient thermocycler. Other methods andapparatus known to those skilled in the art that produce amplicons thatidentify the event MON87705 DNA is within the skill of the art.

DNA amplification in a Stratagene Robocycler, MJ Engine, Perkin-Elmer9700, Eppendorf Mastercycler Gradient thermocycler, Applied BiosystemsGeneAmp PCR System 9700 or MJ Research DNA Engine PTC-225 thermal cycleris performed using the following cycling parameters. When running thePCR in the Eppendorf Mastercycler Gradient or MJ Engine, thethermocycler should be run in the calculated mode. When running the PCRin the Perkin-Elmer 9700, the thermocycler is run with the ramp speedset at maximum.

TABLE 4 Soybean MON87705 Event-Specific Zygosity Endpoint TaqMan PCRStep Reagent Volume Comments 1 18 megohm water adjust for final volumeof 10 μl 2 2X Universal Master Mix   5 μl 1X final concentration 3Zygosity primer - SQ20901, SQ21928, 0.5 μl 1 μM final SQ21905(resuspended in 18 megohm water to concentration a concentration of 20μM for each primer) Example: In a microcentrifuge tube, the followingshould be added to achieve 500 μl at a final concentration of 20 μM: 100μl of each primer at a concentration of 100 μM 200 μl of 18 megohm water4 Event 6-FAM ™ Probe (PB10164) 0.2 μl 0.20 μM (resuspended in 18 megohmwater to a final concentration of 10 μM) concentration Note: 6-FAM ™Probe is light sensitive. 5 Wild Type VIC ™ Probe (PB10335) 0.2 μl 0.20μM final (resuspended in 18 megohm water to a concentrationconcentration of 10 μM) Note: VIC ™ Probe is light sensitive. 6Extracted DNA (template): 3.0 μl Leaf Samples to be analyzed Negativecontrol (non-transgenic DNA) Negative water control (No templatecontrol) Positive control Homozygous GM_A94978 DNA Positive controlHemizygous GM_A94978 DNA

TABLE 5 Zygosity Endpoint TaqMan thermocycler conditions Cycle No.Settings 1 50° C. 2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64°C. 1 minute −1° C./cycle 30 95° C. 15 seconds 54° C. 1 minute 1 10° C.Forever

Example 4 Identification of Event MON87705 in a Given Soybean Sample

The following example describes how one may identify the presence orabsence of MON87705 event in a given soybean sample.

DNA event primer pairs are used to produce an amplicon diagnostic forsoybean event MON87705. An amplicon diagnostic for MON87705 comprises atleast one junction sequence: SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:18. SEQ ID NO: 1 (FIG. 2) is a nucleotide sequence corresponding to thejunction of the 5′ flanking sequence (positions 3413 through 3422 of SEQID NO: 6, see FIG. 2) and the integrated border of the insertion(positions 3423 through 3433 of SEQ ID NO: 6, see FIG. 2). SEQ ID NO: 2(see FIG. 2) is a nucleotide sequence corresponding to the junction ofthe integrated border of the insertion (positions 10664 through 10673 ofSEQ ID NO: 6, see FIG. 2) and the 3′ flanking sequence (positions 10674through 10683 of SEQ ID NO: 6, see FIG. 2). SEQ ID NO: 18 is anucleotide sequence corresponding to the right border to right borderjunction (positions 9230 to 9335 of SEQ ID NO: 6) generated upon thecointegration of the two pMON95829 T-DNAs to yield event MON87705

Event primer pairs that will produce a diagnostic amplicon for MON87705include primer pairs based upon the flanking sequences and the insertedcassettes. To acquire a diagnostic amplicon in which at least 11nucleotides of SEQ ID NO: 1 is found, one would design a forward primerbased upon SEQ ID NO: 3 from bases 1 through 3448 and a reverse primerbased upon the inserted cassette, SEQ ID NO: 5 from positions 10 through7251. To acquire a diagnostic amplicon in which at least 11 nucleotidesof SEQ ID NO: 2 is found, one would design a forward primer based uponthe inserted cassette, SEQ ID NO: 5, from positions 1 through 7241 and areverse primer based upon the 3′ flanking sequence, SEQ ID NO: 4, frombases 10 through 2515. For practical purposes, one should design primerswhich produce amplicons of a limited size range, preferably between 200to 1000 bases. Smaller sized amplicons in general are more reliablyproduced in PCR reactions, allow for shorter cycle times and can beeasily separated and visualized on agarose gels or adapted for use inendpoint TaqMan-like assays. In addition, amplicons produced using saidprimer pairs can be cloned into vectors, propagated, isolated andsequenced or can be sequenced directly with methods well established inthe art. Any primer pair derived from the combination of SEQ ID NO: 3and SEQ ID NO: 5 or the combination of SEQ ID NO: 4 and SEQ ID NO: 5that are useful in a DNA amplification method to produce an amplicondiagnostic for MON87705 or progeny thereof is an aspect of the presentinvention. Any single isolated DNA polynucleotide primer moleculecomprising at least 11 contiguous nucleotides of SEQ ID NO: 3, or itscomplement that is useful in a DNA amplification method to produce anamplicon diagnostic for MON87705 or progeny thereof is an aspect of thepresent invention. Any single isolated DNA polynucleotide primermolecule comprising at least 11 contiguous nucleotides of SEQ ID NO: 4,or its complement that is useful in a DNA amplification method toproduce an amplicon diagnostic for MON87705 or progeny thereof is anaspect of the present invention. Any single isolated DNA polynucleotideprimer molecule comprising at least 11 contiguous nucleotides of SEQ IDNO: 5, or its complement that is useful in a DNA amplification method toproduce an amplicon diagnostic for MON87705 or progeny thereof is anaspect of the present invention.

An example of the amplification conditions for this analysis isillustrated in Table 6 and Table 7. However, any modification of thesemethods or the use of DNA primers homologous or complementary to SEQ IDNO: 3 or SEQ ID NO: 4 or DNA sequences of the genetic elements containedin the transgene insert (SEQ ID NO: 5) of MON87705 that produce anamplicon diagnostic for MON87705, is within the art. A diagnosticamplicon comprises a DNA molecule homologous or complementary to atleast one transgene/genomic junction DNA listed under SEQ ID NO: 1, 2and 18, or a substantial portion thereof.

An analysis for event MON87705 plant tissue sample should include apositive tissue control from event MON87705, a negative control from asoybean plant that is not event MON87705, for example, but not limitedto A3525, and a negative control that contains no soybean genomic DNA. Aprimer pair that will amplify an endogenous soybean DNA molecule willserve as an internal control for the DNA amplification conditions.Additional primer sequences can be selected from SEQ ID NO: 3, SEQ IDNO: 4, or SEQ ID NO: 5 by those skilled in the art of DNA amplificationmethods, and conditions selected for the production of an amplicon bythe methods shown in Table 6 and Table 7 may differ, but result in anamplicon diagnostic for event MON87705 DNA. The use of these DNA primersequences with modifications to the methods of Table 6 and Table 7 arewithin the scope of the invention. The amplicon produced by at least oneDNA primer sequence derived from SEQ ID NO: 3, SEQ ID NO: 4 or SEQ IDNO: 5 that is diagnostic for MON87705 is an aspect of the invention.

DNA detection kits that contain at least one DNA primer derived from SEQID NO: 3,

SEQ ID NO: 4 or SEQ ID NO: 5, that when used in a DNA amplificationmethod, produces a diagnostic amplicon for MON87705 or its progeny is anaspect of the invention. A soybean plant or seed, wherein its genomewill produce an amplicon diagnostic for MON87705 when tested in a DNAamplification method is an aspect of the invention. The assay for theMON87705 amplicon can be performed by using an Applied BiosystemsGeneAmp PCR System 9700, Stratagene Robocycler, MJ Engine, Perkin-Elmer9700 or Eppendorf Mastercycler Gradient thermocycler or any otheramplification system that can be used to produce an amplicon diagnosticof MON87705 as shown in Table 7.

TABLE 6 Soybean MON87705 Event Specific PCR Assay Step Reagent VolumeComments 1 18 megohm water adjust for final volume of 10 ul 2 2XUniversal Master Mix 5.0 ul 1X final concentration (Contains dNTPs,enzyme and buffer) of dNTPs, enzyme and buffer 3 Primer-1 and Primer-2Mix (resuspended in 18 0.5 ul 1.0 uM final megohm water to aconcentration of 20 uM for concentration each primer) Example: In amicrocentrifuge tube, the following should be added to achieve 500 ul ata final concentration of 20uM: 100 ul of Primer 1 at a concentration of100 uM 100 ul of Primer 2 at a concentration of 100 uM 300 ul of 18megohm water 5 Extracted DNA (template) 50 ng of genomic 3.0 ul DNA:Leaf samples to be analyzed Negative control (non-transgenic DNA)Negative water control (no template control) Positive control MON88769DNA

TABLE 7 Soybean MON87705 Event Thermocycler Conditions Cycle No.Settings 1 50° C. 2 minutes 1 95° C. 10 minutes 10 95° C. 15 seconds 64°C. 1 minute −1° C./cycle 30 95° C. 15 seconds 54° C. 1 minute 1 10° C.Forever

A deposit of seeds of the soybean event MON87705 disclosed above andrecited in the claims, has been made under the Budapest Treaty with theAmerican Type Culture Collection (ATCC), 10801 University Boulevard,Manassas, Va. 20110. The ATCC accession number is PTA-9241. The depositwill be maintained in the depository for a period of 30 years, or 5years after the last request, or for the effective life of the patent,whichever is longer, and will be replaced as necessary during thatperiod.

Having illustrated and described the principles of the presentinvention, it should be apparent to persons skilled in the art that theinvention can be modified in arrangement and detail without departingfrom such principles. We claim all modifications that are within thespirit and scope of the appended claims.

1. A soybean plant comprising a DNA molecule comprising a polynucleotidehaving a sequence that is or is complementary to two or more sequencesselected from the group consisting of SEQ ID NOs: 1, 2 and
 18. 2. Aplant part of the soybean plant of claim 1, wherein said plant partcomprises a polynucleotide having a sequence that is or is complementaryto two or more sequences selected from the group consisting of SEQ IDNOs: 1, 2 and
 18. 3. Progeny of the soybean plant of claim 1, whereinsaid progeny comprises a polynucleotide having a sequence that is or iscomplementary to two or more sequences selected from the groupconsisting of SEQ ID NOs: 1, 2 and
 18. 4.-8. (canceled)
 9. A DNAmolecule comprising a polynucleotide having a sequence that is or iscomplementary to one selected from the group consisting of sequenceslisted under SEQ ID NOs: 2 and
 18. 10. An isolated DNA moleculecomprising at least from about 11 to about 20 consecutive nucleotidesselected from SEQ ID NO: 1 or SEQ ID NO:
 2. 11. A genome of a soybeancell comprising a polynucleotide having a sequence selected from thegroup consisting of two or more sequences listed under SEQ ID NOs: 1, 2and
 18. 12.-21. (canceled)
 22. A composition having a DNA moleculeconsisting of two or more sequences selected from the group consistingof SEQ ID NOs: 1, 2 and 18, wherein said composition is a commodityproduct selected from the group consisting of soybean meal, soy flour,soy protein concentrate, soy protein isolates, texturized soy proteinconcentrate, hydrolyzed soy protein and whipped topping.
 23. A commodityproduct obtained from a soybean plant of claim 1, wherein said commodityproduct is selected from the group consisting of soybean meal, soyflour, soy protein concentrate, soy protein isolates, texturized soyprotein concentrate, hydrolyzed soy protein, whipped topping, whole soyseed, processed soy seed, animal feed, soymilk, soy nut butter, natto,tempeh, edible raw whole soybean pods, roasted whole soybean pods,edamame, soymilk, soy yogurt, soy cheese, tofu, yuba, and biomass.
 24. Acommodity product comprising the composition of claim 22, wherein saidcommodity product is selected from the group consisting of soybean meal,soy flour, soy protein concentrate, soy protein isolates, texturized soyprotein concentrate, hydrolyzed soy protein, whipped topping, whole soyseed, processed soy seeds, animal feed, soymilk, soy nut butter, natto,tempeh, edible raw whole soybean pods, roasted whole soybean pods,edamame, soymilk, soy yogurt, soy cheese, tofu, yuba, and biomass. 25.The isolated DNA molecule of claim 10, comprising at least from about 11to about 20 consecutive nucleotides selected from SEQ ID NO:
 1. 26. Theisolated DNA molecule of claim 10, comprising at least from about 11 toabout 20 consecutive nucleotides selected from SEQ ID NO:
 2. 27. A DNAamplicon comprising a polynucleotide having a sequence that is, or iscomplementary to, a sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
 18. 28. The amplicon of claim 27,wherein said amplicon comprises a sequence SEQ ID NO: 1, SEQ ID NO: 2,and SEQ ID NO:
 18. 29. The amplicon of claim 27, wherein said ampliconcomprises SEQ ID NO:
 1. 30. The amplicon of claim 27, wherein saidamplicon comprises SEQ ID NO:
 2. 31. The amplicon of claim 27, whereinsaid amplicon comprises SEQ ID NO: 18.